Antibiotic 19,402 r.p.

ABSTRACT

The new antibiotic designated 19,402 R.P. - an acid - is obtained by the cultivation of the microorganism &#39;&#39;&#39;&#39;Streptomyces 6227&#39;&#39;&#39;&#39; or &#39;&#39;&#39;&#39;Streptomyces peruviensis&#39;&#39;&#39;&#39; NRRL 2757, the characteristics of which are described in British Pat. No. 846,801. The antibiotic and its alkali metal salts are particularly active against gram-positive microorganisms.

United States Patent [191 Mancy et al.

[ ANTIBIOTIC 19,402 RP.

[75] Inventors: Denise Mancy, Val-de-Marne; Leon Ninet; Jean PreudHomme,both of Paris, all of France [73] Assignee: Rhone-Poulenc S.A., Paris,France [22] Filed: Feb. 21, 1968 21 Appl. No.: 707,233

[30] Foreign Application Priority Data Feb. 22, 1967 France 67.96051[52] US. Cl. 424/117, 195/80 [51] Int. Cl A6lk 21/00 [58] Field ofSearch 424/117; 195/80 [56] References Cited FOREIGN PATENTS ORAPPLICATIONS 846,801 8/1960 Great Britain...- 424/117 in km [1113,821,366 [4 June 28, 1974 Primary Examiner-Jerome D. Goldberg Attorney,Agent, or Firm-Stevens, Davis, Miller and Web [ ABSTRACT The newantibiotic designated 19,402 R.P. an acid is obtained by the cultivationof the microorganism Streptomyces 6227 or Streptomyces peruviensis" NRRL2757, the characteristics of which are described in British Pat. No.846,801. The antibiotic and its alkali metal salts are particularlyactive against gram-positive microorganisms.

2 Claims, 1 Drawing Figure ANTIBIOTIC 19,402 R.P.

Thisinvention relates to new antibiotic, hereinafter designated by theNo. 19,402 R.P., and salts thereof, to a process for their preparationand pharmaceutical compositions containing them.

This new antibiotic is of very particular interest because of its highantibacterial activity against grampositive microorganisms.

19,402 RF. is obtained from artificial culture media containing amicroorganism belonging to the streptomyces genus and designated by thename Streptomyces 6227 or Streptomyces peruviensis (NRRL 2757), whichhas already been described in British Pat. No. 846,801 entitled-Antiobiotic 6798 RF. and process for its preparation applied for on May27, 1958 and granted to Societe des Usines Chimiques Rhone-Poulenc. Thedescription of the culture characteristics and biochemical properties ofSlreptomyces 6227 or Slreptomyces peruviensis (NRRL 2757), a culture ofwhich is with the Northern Utilization Research and Development Divisionof the Agricultural Research Service of the United States Department ofAgriculture at Peoria, 111., and assigned the No. NRRL 2757, in thatpatent is incorporated in this specification by specific reference.

19,402 RF. is an acid whose sodium salt has the followingphysico-chemical properties:

Appearance: white amorphous powder Solubility: very soluble in water,soluble in anhydrous methanol, and in ethanol, propanol and isopropanolcontaining water, very sparingly soluble in anhydrous ethanol, propanol,isopropanol and butanol, and insoluble in acetone, hexane, benzene,diethyl ether, chloroform and ethyl acetate. Ultra-violet spectrum(determined with a solution of 0.04 mg/l. in water):

absorption maximum at 256 nm E 102.5

absorption minimum at 227 nm E, 18.7

TABLE I 3420 vs 1620 m i 1070 vs 710 sh where 3300 sh 1530 s 1045 s 665111 vs very strong 3100 sh 1420 m 972 m 595 w s strong 2930 s 1380 m 955w 575 s m medium 2870 m 1330 m 883 m 495 m w weak 1720 m 1240 m 855 m475 sh sh shoulder 1675 s 1155 sh 800 w 425 w 1645 m 1101) s 775 w3li5'nh Optical rotation [01],, +1.9 1 (c 0.4, water) 1 [a],;,,, 7.5:1'(c 0.4, water) [011 9,051" (c 0.4, water) Elementary composition C 46.25percent, H 6.9 percent, 0 36.7 percent, N 4.1 percent, P 2.4 percent, S0.6 percent, Na 3.05 percent.

It does not dialyse across a regenerated cellulose membrane (Cellophanetype).

The sodium salt of 19,402 R.P. can be identified by electrophoreticmigration on an agarose gel buffered to pH 8 by means of a glycinebuffer (agarose is a neutral linear polymer of galactose): there is amigration of 13 X 10 cm/volt/hour towards the anode.

The sodium salt of 19,402 R.P. can also be identified by chromatographyon various supports. The Rf values obtained with various developmentsolvents are given in Table ll (development is carried out at 20C., andmicrobiological revelation by applying the chromatograms to an agarplate inoculated with Staphylococcus aureus 209 P for 5 minutes and thenincubating overnight at 37C.).

(50:40:30 by volume) The bacteriostatic activity of 19,402 R.P. towardsa certain number of microorganisms was determined by a conventionaldilution method. For each microorganism the minimum concentration ofantibiotic was determined which, under specified conditions, inhibitsany visible development in an appropriate nutrient broth. The results ofvarious determinations are summarised in Table Ill, in which the minimumbacteriostatic concentrations of the antibiotic are expressed inmicrograms of substance per cc. of test medium.

These various determinations show that the activity of 19,402 RF. isprincipally exerted against microor ganisms which accept the Gram stain;its activity against Streptococcus haemolyticus is particularly high. Ithas relatively little effect on gram-negative microorganisms althoughits activity against Neisseria gonorrhaeae and Brucella abortus bovis isappreciable. It does not show any cross resistance with the followingantibiotics: penicillin, streptomycin, tetracycline, chloramphenicol,spiramycin, carbomy'cin, erythromycin, pristinamycin and novobiocin.

The antibacterial activity of 19,402 R1. was confirmed in vivo withlaboratory animals experimentally infected with microorganisms such asstreptococci, pneumococci and staphylococci. It proved particularlyeffective when administered subcutaneously to mice. It also possesses avery good preventive activity towards staphylococcal or streptococcalinfections of mice when administered subcutaneously or intravenously.

The toxicity of 19,402 R.P. has been studied principally with mice. The50 percezr tlethal dose, or L D TABLE III Bacterial organisms testedMinimum bacteriostatic concentrations (in pglcc.)

Stu 1h ylnmrzus aura m I33 strain (institut Pasteur) Slap/1 ylut'uu'usuureus Smith strain Sart'ina Iulea ATCC 934i Slreplm'm'cus faet'alisATCC 9790 Slreplmm'cus viridanr (Institut Pasteur) Slreplomccus pyngeneshaemolylic'us (Dig 7 strain.

Institut Pasteur) Neisseria gonorrhaeae (A I50 Institut Pasteur)Diplomat-us pneumoniae (Til strain, Institut Pasteur) Bacillus sublilisATCC 6633 Bacillus cereus ATCC 6630 Mycobacrerium species ATCC 607Mycobarrerium para smcgmalis (A 75 Lausanne) Escherichia call ATCC 9637Shigella dysenteriae Shiga L (lnstitut Pasteur) Salmonella paratyphi A(Lacasse, lnstitut Pasteur) Salmonella scholtmuelleri (paratyphi B)Fougenc (Institut Pasteur) Proteus vulgaris Klebsiella pneumoniae ATCC10,031

Pseudamonas aeruginora (Bass strain lnstitut Pasteur) BruceIIabronchiseprica (CN 387 Wellcome Institute) Brucella abortus bovis B 19Pasleurella multocida (A .125, lnstitut Pasteur) Reiters treponema wasdetermined intraperitoneally, and found to be 600 mg./kg. This resultshows that the antibiotic is of very low toxicity.

According to a feature of the invention, a process for the production of19,402 R.P. comprises inoculating an aqueous nutrient medium, containingassimilable sources of carbon, nitrogen, and mineral salts, with aculture of the strain Streptomyces peruviensis, or a 19,402 R.P.producing mutant thereof, allowing aerobic fermentation to take placeuntil a substantial amount of 19,402 R.P. is produced by the saidmicroorganism in the said culture medium and isolating 19,402 R.P. assuch, or an alkali metal salt thereof, from the culture medium.

The source of carbon may comprise carbohydrates such as glucose,sucrose, dextrose, starch, lactose, or molasses; or sugar-alcohols,especially mannitol and glycerin. However, animal or vegetable oils suchas lard or soya bean oil may also be employed with advantage.

Suitable sources of nitrogen for the fermentation may be extremelyvaried. They may be simple mineral or organic salts of ammonia such asthe chloride, sulphate, nitrate, phosphate, acetate, lactate, citrate,and tartrate. There may also be employed much more complex substances ofanimal or vegetable origin such as meat extracts, fish flours, soya beanmeal, peanut flour, corn-steep, extracts and autolysates of yeast, andcasein hydrolysates.

Among the mineral elements employed some have a buffering effect such ascalcium or magnesium carbonate and alkali or alkaline earth metalphosphates. Others afford the ionic equilibrium necessary for theoptimum production of the antibiotic such as the chlorides or sulphatesof alkali or alkaline earth metals. Finally, some have a more specificaction upon the metabolic reactions of Streptomyces peruviensis. Theseare the salts of zinc, cobalt, copper, manganese and iron.

The pH of the fermentation medium at the start of yl ytei gi lsilzefl-flanstltilh mttm fermentation temperature is about 30C. but asatisfactory yield is obtained with temperatures between 25 and 35C.Aeration of the fermentation liquors is not a critical factor, but,nevertheless, aerations of 0.5 to 2 liters of air per liter of broth perminute are particularly suitable. Very good yields of 19,402 R.P. areobtained after about 5 days of culture.

The fermentative growth of Streptomyces peruviensis may be carried outby surface-culture technique, but submerged culture methods followingthe techniques which are commonly used for this kind of fermentation arepreferred.

These conditions for the culture of Streptomyces peruviensis for theproduction of 19,402 R.P. are similar to the conditions described inBritish Pat. No. 846,801 for the production of the antibiotic 6,798R.P., which is an amphoteric substance, also obtained by cultivation ofthe same strain of streptomyces.

19,402 R.P. is isolated from the fermentation broths by conventionalmethods for the extraction and purification of acid non-dialysableantibiotics, for example as described in British Pat. No. 1,086,780entitled New Antibiotic applied for on November 16, i965 and granted toRhone-Poulenc SA.

The fermentation broth may be filtered at a pH greater than or equal to5, preferably between 7 and 9, but under these conditions a large partof the 19,402 R.P. remains in the filtration cake which also has to betreated in order to extract the desired antibiotic. F urtherrnore, it isthen necessary to separate 19,402 R.P.

from the antibiotic 6,798 R.P., which has also been produced during thecultivation of Streptomyces peruviensis and has also passed into thefiltrate.

most five carbon atoms, such as methanol, ethanol or propanol.

The fermentation broth may also be passed through a column containing anion exchange resin of strongly anionic character and high porosity, andthe exchanger then eluted with an aqueous-alcoholic solvent, forexample, aqueous methanol, containing an electrolyte. This method alsosuffers from the disadvantage of requiring a subsequent separation of19,402 R.P. from 6,798 R.P.

Crude 19,402 R.P. may be isolated from the aqueous-alcoholic solutionsindicated above by concentrating the solution to a small volume; thisconcentration is conveniently. carried out at a temperature below 40C.under reduced pressure. The crude antibiotic, or if desired one of itsalkali metal salts, precipates on cooling and/or on addition of a poorsolvent for 19,402 R.P. or its alkali metal salts, for example,anhydrous ethanol or acetone.

19,402 R.P. can then be purified by fixing it on an ion exchange resinof strongly anionic character and of high porosity and then eluting withan aqueousalcoholic solution containing an electrolyte such as sodium,ammonium, potassium, calcium or magnesium chloride at a concentration of5 to 50 g. per liter of eluant. The eluate is then concentrated to asmall volume at a temperature below 40C. and under reduced pressure, andthe concentrate is dialysed against a stream of distilled water by meansof a regenerated cellulose membrane. The inorganic salts and variousimpurities are entrained by the water and 19,402 R.P. remains completelyin the dialysed solution. The purified antibiotic 19,402 R.P. isobtained by lyophilising this solution.

In order to obtain ever purer 19,402 R.P., conventional methods may beemployed, such as chromatog raphy on various absorbents, countercurrentdistribution or partition between various solvents. It has provedespecially advantageous to chromatograph the antibiotic in aqueoussolution on silica gel and to elute with a mixture of propanol andammonia.

It will be understood that the various methods referred to above for theextraction, isolation and purification of 19,402 R.P. may be repeatedseveral times as required for the production of this antibiotic in aform appropriate for the envisaged application.

After these various treatments 19,402 R.P. is preferably obtained in theform of an alkali metal salt. Such a salt may thereafter, if desired, beconverted to the free acid by preparing a concentrated solution of thealkali metal salt in water and passing this over a strongly cationic ionexchange resin.

The following non-limitative examples illustrate the invention. In thefollowing the activity is always determined by biological determinationby the diffusion method, using Bacillus subtilis as the sensitivemicroorganism and with reference to a pure sample of 19,402 R.P. asstandard. This activity is expressed in units per mg. for solidproducts'and in units per cc. for solutions. The unit (abbreviated to u)is the minimum quantity of product which, when dissolved in 1 cc. of asuitable culture medium, inhibits the growth of Staphylococcus aurus 209under a particular set of conditions.

EXAMPLE I A l70-liter fermentation vessel is cerelose 2.400 kg.

sodium chloride 0.600 kg.

magnesium sulphate 0.120 kg.

water to make up to 1 10 liters After having adjusted the pH of themixture to 7.40 by-means of concentrated sodium hydroxide solution (dl.33;660 cc.), calcium carbonate (0.600 kg.) is added. The culturemedium is then sterilised by bubbling steam at 122C. through it for 40minutes. After cooling, the volume of the broth is 120 liters and the pHis 6.95. The medium is then inoculated with a culture (200 cc.) of thestrain Streptomyces peruviensis in a stirred Erlenmeyer fiask. Theculture is developed at C for 26 hours with stirring and aerationwithste rile air; it is then ready for inoculation of the productionculture.

The production culture is carried out in an 800-liter fermentationvessel charged with the following sub stances:

corn steep 22 kg starch 8.250 kg.

soya oil 8.250 1.

calcium carbonate 5.500 kg.

monopotassium phosphate 1.100 kg.

magnesium sulphate 1.100 kg.

hydrated cobalt chloride 11 g.

water to make up to 510 litres.

The medium is sterilised by bubbling steam at 122C through it for 40minutes. After cooling, the volume of the broth is 550 liters and the pHis 6.60 after addition of concentrated sodium hydroxide solution (d1.33;250 cc.). The broth is then inoculated with the inoculum cultureliters) from the l-literfermentation vessel mentioned above. Theproduction culture is carried out at 30C for 123 hours with agitation,using a motor rotating at 285 rpm and aeration with 25 m /hour ofsterile air. The pH of the medium is then 8.1 and the volume of thebroth is 520 liters. The amount of antibiotic present in thefermentation broth is 2420 u/cc.

EXAMPLE ii The fermentation broth obtained by the procedure of Example I(520 liters), of strength 2420 u/cc. and at pH 8.1, is adjustedto pH 4by adding a 37 percent strength solution of phosphoric acid in a vatprovided with a stirrer. A filtration aid (15 kg.) is then added. Themixture is filtered on a filter press and the filter cake is washed withtapwater (250 liters). The filtrate and the practically inactive washwater are discarded down the drain. The filter cake is suspended, withstirring, in a mixture (350 liters) consisting of methanol (270 liters)with the complement being .water. The apparent pH of the mixture is thenadjusted to 7 by adding a l0N sodium hydroxide solution. Stirring iscontinued for 1 hour, and the sludge is then filtered on a filter press.The filtrate is collected and the cake is washed with a watermethanolmixture liters) containing 70 percent of methanol. The combined filtrateand wash liquid (amounting to 440 liters) has a strength of 2,320 u/cc.The cake is discarded.

The methanolic filtrate is concentrated under reduced pressure (35mm.Hg) at 37C to a volume of 6 liters, and ethanol (3 liters) is addedto the concentrate. The antibiotic in the resulting solution is thenprecipitatedby means of a mixture of ethanol (30 liters) and acetone (40liters). The antibiotic is isolated by filtration, washed with acetoneand dried in an oven in vacuo mm.Hg). A brown product (1,009 g.) is thusobtained having a strength of 950 u/mg; this is the crude sodium salt of19,402 R.P.

EXAMPLE Ill The crude product (900 g.) prepared as described in Example11 (of strength 950 u/mg.) is dissolved in distilled water liters). Thesolution is filtered and then passed through a column (internal diameter9 cm) containing 8 liters of Dowex 1X2 resin in chloride form (flow rateabout 2 liters per hour). The column is successively washed with:

distilled water until the effluent is colorless 8 l. at

2 l/hour formic acid water mixture (10 90 by volume) 24 l. at 12 l/hourformic acid water-methanol mixture (10 l0 80 by volume) 24 l. at 12l/hour methanol water mixture (80 by volume) 24 l. at 12 l/hour.

The antibiotic is then eluted with a mixture of methanol and water (80 z20 by volume) to which 10 g/l. of potassium chloride have been added,using a flow rate of about 10 l./hour. The eluate is divided intofractions every 10 liters; the most active fractions (4,5 and 6) arerecombined and concentrated under reduced pressure, at a temperaturebelow 40C, to 3 liters.

The concentrate is dialysed for 48 hours against distilled water (threetimes 40 liters) through a regenerated cellulose membrane to remove thesalts and various impurities and is then lyophilized.

The potassium salt of 19,402 R.P. (35 g.) is obtained in the form of abeige amorphous powder of strength 13,800 u/mg, having an absorptionmaximum in the ultra-violet at 257 nm (E 84).

EXAMPLE 1V The purified antibiotic obtained as described in Example 111(5 g.) is dissolved in distilled water (20 cc.) and worked into a pastewith activated silica gel (25 g.); the resulting paste is driedovernight under a pressure of 2 mm.Hg at ambient temperature and thenintroduced into the upper part of a column (internal diameter 4 cm)previously charged with dry activated sil ica gel (500 g.).

The column is successively developed with the following mixtures:

n-propanol and 2N ammonia 100:20 by volume) 2.4 liters n-propanol and 2Nammonia (80:20 by volume) 2 liters and then eluted with a mixture ofnpropanol and 2N ammonia (80:30 by volume), dividing into fractionsevery 50 cc.

Fractions 1 to 50 on the one hand, and 51 to 75 on the other hand, arerecombined, concentrated under reduced pressure at a temperature below40C in order to drive off the propanol, and then lyophilised.

Fractions 1 to 50 thus yield:

crop a 1,300 mg. of strength 15,700 u/mg. 2

Fractions 51 to 75 thus yield:

crop B: 1,355 mg. of strength 15,800 u/mg.

Crop A 1,200 mg.) is dissolved in distilled water (20 cc.) and thesolution added, with stirring and in small fractions, to Amberlite [R120 resin in the acid state until a constant pH is obtained. The resinis filtered and rinsed with distilled water (10 cc.). The filtrate andwash liquids are combined, adjusted to pH 8.0 with sodium hydroxidesolution and dialysed overnight against distilled water (twice 1 liter)through a regenerated cellulose membrane. The sodium salt of 19,402 R.P.is obtained in the solid form by lyophilising the solution which remainsinside the membrane.

The pure sodium salt of 19,402 R.P. (945 mg). of strength 17,800 u/mg,is thus obtained; it has the following elementary composition:

C 46.25 percent, H 6.9 percent, 0 36.7 percent, N 4.1 percent, P 24percent, S 0.6 percent, Na 3.05 percent.

The present invention also includes within its scope pharmaceuticalcompositions comprising 19,402 R.P., or a non-toxic salt thereof(preferably an alkali metal salt), in association with apharmaceutically acceptable carrier, which may itself be physiologicallyactive. Such compositions may be in any pharmaceutical form suitable forthe method of administration envisaged.

The proportion of 19,402 R.P. in these pharmaceutical compositions willvary according to the desired therapeutic effect. For treatinginfections by Grampositive microorganisms intramuscularly orintravenously, the dose is generally between 0.25 and 1.5 g. for anadult. This dose may be repeated eight days later if need be.

The following example illustrates pharmaceutical compositions accordingto the invention.

EXAMPLE V A solution of the following composition is prepared:

sodium salt of 19,402 R.P. 50 g. distilled water sufficient to make upto 500 cc.

This solution is sterilised by filtration through a bacteriostaticfilter and is then divided between ampoules, introducing 5 cc. into eachampoule. The contents of the ampoules are lyophilised and then theampoules are sealed. In order to use the antibiotic in these ampoulesparenterally, an injectable solution is prepared immediately before useby adding distilled water (5 cc.) to the lyophilised contents of theampoule. A solution (about 5 cc.) containing the active principle (0.5g.) is then obtained.

We claim:

1. The antibiotic herein designated 19,402 R.P. in the form of thesodium or potassium salt, its sodium salt having the followingcharacteristics:

a white amorphous powder which is very soluble in water, soluble inanhydrous methanol, and in ethanol, propanol and isopropanol containingwater, very sparingly soluble in anhydrous ethanol, propanol,isopropanol and butanol, and insoluble in acetone, hexane benzene,diethyl ether, chloroform and ethyl acetate; it has the elementarycompositlon C 46.25 percent, H 6.9 percent, 0 36.7 percent. N 4.1percent, P 2.4 percent, S 0.6 percent, Na 3.05%; its optical rotation[01],, 19 i 1 (C 0.4, water) [M 7.5 i 1 (C 0.4, water), and [M 9 i 1 (C0.4, water) its ultra-violet spectrum (determined with a solution of0.04 mg./l. in water) shows an absorption maximum at 256 nm (E 102.5)and an absorption minimum at 227 nm (E 18.7), and its infra-red spectrum(determined with tablets of a mixture with potassium bromide) showsprincipal absorption bands as follows: 3420 very strong, 3300 shoulder,3100 shoulder, 2930 strong, 2870 medium, 1720 medium, 1675 strong, 1645medium, 1620 medium, 1530 strong, 1420 medium, 1380 medium, 1330 medium,1240 medium, 1155 shoulder, 1100 strong, 1070 very strong, 1045 strong,972 medium, 955 weak, 883 medium, 855 medium, 800 weak, 775 weak, 710

tion with a phannaceutically acceptable carrier.

2. Pharmaceutical compositions which comprise, as active ingredient anantibacerially effective amount of the sodium or potassium salt of19,402 R.P. in association with a pharmaceutically acceptable carrier.